CD80 and CD86 Binding Protein Compositions and Uses Thereof

ABSTRACT

This invention relates to CTLA-4 protein compositions and their use in the mitigation of autoimmune adverse events associated with cancer immunotherapy.

BACKGROUND OF THE INVENTION

Treatment with anti-CTLA-4 antibodies has been shown to be a powerfultool for enhancing anti-tumor immunity in preclinical models (10).Monotherapy with an antibody against CTLA-4 promoted rejection oftransplantable tumors of various origins. Based on promising preclinicaltumor model studies, the clinical potential of antibodies against CTLA-4has been explored in different human malignancies. Although anti-CTLA-4marketed as Yervoy, disclosed in U.S. Pat. No. 6,984,720) hasdemonstrated efficacy in treating melanoma, treatment and targeting ofCTLA-4 is associated with autoimmune-like toxicities. In addition,anti-CTLA-4 mAbs such as Ipilimumab and Tremelimumab are used incombination therapy with anti-PD-1/PD-L1 antibodies with superiortherapeutic effect. However, the improved therapeutic effect isassociated even higher rates of grade 3 and grade 4 organ toxicity.Characteristic side effects from inhibition of CTLA-4 are generallycalled immune-related adverse events (irAEs) and the most common irAEsare skin rash, hepatitis, colitis and endocrinopathies, particularlyhypopituitarism. Therefore, there is a large unmet medical need to treatirAE while preserving the cancer therapeutic effect of anti-CTLA-4monoclonal antibody (mAb).

The inventors have demonstrated that both clinically proven therapeuticanti-human CTLA-4 mAb and two anti-mouse Ctla-4 mAbs induce tumorrejection without blocking B7-CTLA-4 interactions under physiologicallyrelevant conditions. Therefore, such blockade was not necessary fortumor rejection even for the mAb that can potently block B7-CTLA-4interactions. These data refine the hypothesis that anti-CTLA-4 mAbconfers an immunotherapeutic effect through checkpoint blockade (108).In support of this notion, it has further been shown that theimmunotherapeutic effect mediated by Ipilimumab and possibly otheranti-CTLA-4 mAbs, was unaffected by blocking B7-1 and B7-2, which iscritical for pathogenesis of autoimmune diseases.

Accumulating data demonstrated that the human CTLA-4 gene encodes twodifferent isoforms of proteins through alternative splicing: one with atrans-membrane domain which is thus likely to be anchored in membrane,and another that lacks the trans-membrane domain and is predicted to besecreted (sCTLA-4) (128). Importantly, genetic studies demonstrated thata polymorphism of CTLA-4 that reduces the relative abundance of thesoluble isoform strongly associates with multiple autoimmune diseases(64). The fact that subjects with autoimmune prone alleles express lesssCTLA-4 mRNA suggests that sCTLA-4 may be protective. This is notion issupported by the broad therapeutic effect of abatacept (129,130), whichis a form of soluble CTLA-4, and by a genetic study in which theselective ablation of the sCTLA-4 isoform accelerated the development oftype I diabetes in the mice (131). Based on these genetic data, theinventors had the insight that an anti-CTLA-4 antibody that shows thepoorest binding to soluble CTLA-4 should give the least irAE. Indeed,based on the impact of the antibodies on the body weight gain in micetreated with anti-CTLA-4 antibodies during the perinatal period, astrong correlation was found among four anti-CTLA-4 mAbs: Ipilimumab hasthe strongest binding for sCTLA-4 and is the most toxic anti-CTLA-4,whereas antibody L3D 10 had the weaker binding to sCTLA-4 and was theleast toxic. Furthermore, humanized L3D10 variants that preferentiallyreduced binding to sCTLA-4 showed further improved the safety profileover the parent antibody.

The protective function of soluble CTLA-4 molecules further support anapproach of using soluble CTLA-4 fusion proteins to mitigate, reduce ortreat irAE. However, since patients with anti-CTLA-4 mAb induced irAEhave circulating anti-CTLA-4 mAbs, CTLA-4 fusion proteins such asabatacept will not only reduce the therapeutic effect of anti-CTLA-4mAbs by preventing them from binding to cell-associated. CTLA-4molecules, but also be rendered ineffective because they will be clearedfrom circulation after forming immune-complex with circulatinganti-CTLA-4 mAbs. Accordingly, there is a need in the art for improvedCTLA-4 immunotherapy compositions and methods.

SUMMARY OF THE INVENTION

This invention relates to human CTLA-4 proteins and anti-B7-1 andanti-B7-2 compositions, and their use for immunotherapy and thetreatment of autoimmune disease and inflammation, and for the reductionof autoimmune side effects associated with anti-CTLA-4 immunotherapy.Specifically, the invention relates to a CTLA-4 protein that exhibitsreduced or eliminated binding to anti-CTLA-4 antibodies used in cancerimmunotherapy but retains the ability to bind to B7-1 and B7-2.

The CTLA-4 protein may comprise a CTLA-4 protein in which the aminoacids of an anti-CTLA-4 antibody binding epitope are modified or mutatedin order to reduce or abolish binding of an anti-CTLA-4 antibody to theCTLA-4 protein, as compared to a wild-type extracellular domain ofCTLA-4. The CTLA-4 protein may have anti-cancer immunotherapeuticactivity. The anti-CTLA-4 antibody may be Ipilimumab. The. CTLA-4protein. may retain its ability to bind at least one of B7-1 and B7-2.The CTLA-4 protein may not block the anti-cancer immunotherapeuticeffects of the anti-CTLA-4 antibody.

The CTLA-4 protein may comprise an extracellular domain of mature humanCTLA-4, a variant thereof, or an. active fragment thereof The CTLA-4protein may comprise an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 34, 36, 38, 39, 40 and 46-48, a variantthereof, and an active fragment thereof. The CTLA-4 protein may besoluble. The CTLA-4 protein may comprise a CTLA-4 fusion protein whereinthe extracellular domain of human CTLA-4 is attached to a humanimmunoglobulin heavy chain constant region. The CTLA-4 protein maycomprise an amino acid sequence selected from the group consisting ofSEQ ID NO: 17, 19, 21, 22, 23, and 42-44. The CTLA-4 protein may bebelatacept.

In another embodiment, the invention relates to antibody compositionsthat bind at least one of human B7-1 and B7-2, and their use forimmunotherapy and the treatment of autoimmune disease and inflammation,and for the reduction of autoimmune side effects associated withanti-CTLA-4 immunotherapy.

Also provided herein is a pharmaceutical composition comprising atherapeutically effective amount of a protein or antibody describedherein. The pharmaceutical composition may comprise a physiologicallyacceptable carder or excipient.

In another aspect, provided herein are methods for reducing one or moreimmune functions or responses in a subject, comprising administering toa subject in need thereof the CTLA-4 protein or anti-B7 antibodycomposition, or a pharmaceutical composition thereof. In a specificembodiment, presented herein are methods for preventing, treating, ormanaging a disease in which it is desirable to inhibit or reduce one ormore immune functions or responses. The disease may be an autoimmunedisease, such as rheumatoid arthritis (RA) or Juvenile IdiopathicArthritis (JIA).

The compositions described herein may also be used to mitigate, minimizeor treat the immune related adverse effects associated withimmunotherapy. In particular, the composition may comprise a moleculethat blocks or reduces the function of B7-1 and B7-2 without affectingthe cancer immunotherapeutic activity of an anti-CTLA-4 antibody. Themolecule may be a CTLA-4 protein, an anti-B7 antibody, or apharmaceutical composition thereof. The molecule may be an antibody thatcan functionally block binding of at least one of B7-1 and B7-2 to atleast one of CD28 and CTLA-4, and may be anti-B7-1 or anti-B7-2. Theantibody may be capable of binding both B7-1 and B7-2, and may comprisea binding site that reacts with both B7-1 and B7-2.

A composition described herein may be administered to a subject, who mayhave cancer, in combination with, or on a background of, anti-CTLA-4immunotherapy. The composition may be used prophylactically to preventirAEs before anti-CTLA-4 treatment is initiated or the before theclinical signs of irAEs emerge. In another embodiment, the compositionis used therapeutically to treat irAEs after anti-CTLA-4 treatment isinitiated and the clinical symptoms are diagnosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. Human CTLA-4 sequence. FIG. 1A shows the sequence of humanCTLA-4 protein (NCBI accession number NP_05205; SEQ ID NO: 1) with thesignal peptide in bold, the IgV domain underlined and the transmembranedomain double underlined. FIG. 1B shows the sequence of abatacept (SEQID NO: 2) with the CTLA-4 sequence shown in bold with the remainder ofthe protein comprising the IgG1 Fc region.

FIG. 2. Therapeutic effect of chimeric L3D10 and 10D1 in the MC38 tumormodel. Human CTLA-4-knock-in mice with body weight of approximately 20grams were used for the study. 1×10⁶ MC38 tumor cells were injectedsubcutaneously into Ctla-4^(h/h) mice and when the tumor reached a sizeof 0.5 cm in diameter, tumor bearing mice were randomized into threegroups with 5 or 6 mice each. Mice were then treated (i.p.) with 100μg/injection of 10D1, chimeric L3D10 or control hIgGFc on days 7, 10,13, and 16 as indicated by the arrows. The results of duplicateexperiments are shown (left and right panels) and data shown are meansand S.D. of tumor size (n=6 per group in the left panel, n=5 per groupin the right panel). L3D10 and 10D1 have similar therapeutic effect inthis model and are both able to induce complete remission of establishedtumors. The diameters (d) of the tumors were calculated using thefollowing formula: D=√(ab), V=ab2/2, where a is the long diameter, whileb is the short diameter. Statistical analyses were performed by two-wayrepeated measures ANOVA (treatment X time). For the left panel: P=10D1vs. hIgGFc: 5.71e-07; L3D10 vs. hIgGFc: P=5.53e-07; 10D1 vs. L3D10:P=0.869.

FIG. 3. Adverse effects of chimeric L3D10 and 10D1 in combination withanti-PD-1. Top panel depicts the experimental design. 10-day oldfemale-only human CTLA-4-knockin mice with body weight of greater than 4grams were used for the study. They received indicated proteins or theircombinations. Arrows indicate time of treatment (100 μg/mice/treatment).Data shown are means and S.D. of % weight gains. Chimeric L3D10 and 10D1have comparable cancer therapeutic effect in adult mice (FIG. 2) butdistinct adverse effects are seen when 10D1 is combined with theanti-PD-1 mAb.

FIG. 4. Adverse effects of chimeric L3D10 and 10D1 in combination withanti-PD-1. The graph shows the terminal body weight on Day 42 in themice from the experiment outlined in FIG. 3 that received either controlIgG, 10D1+anti-PD-1 or chimeric L3D10+anti-PD-1 (n=5 per group). Asignificant reduction in weight is observed with the anti-PD1+10D1combination, which was not seen with the anti-PD-1+Chimeric L3D10combination.

FIG. 5. Pathological effects of chimeric L3D10 and 10D1 in combinationwith anti-PD-1. Group 1 is hIgG, Group 2 is 10D1+anti-PD1, and Group 3is L3D10+anti-PD1. To further examine to relative toxicity of L3D10compared to 10D1 when administered in combination with anti-PD-1, thegross anatomy of the mice described in FIG. 4 above was investigated.The Uterus/Ovary/Bladder and thymus were noticably smaller in in icetreated with 10D1+PD-1, whereas the organs in mice treated withL3D10+anti-PD-1 was comparable to hIgG control. In contrast, the heartsdissected from mice treated with 10D1 appeared larger in size with anoticeably whiter appearance.

FIGS. 6A-D. Treatment with 10D1 in combination with anti-PD-1 results inabnormal erythropoiesis. Given the differences in the hearts observed inFIG. 5, erythropoiesis was investigated within the mice and deardifferences were observed in the mice treated with 10D1+anti-PD-1relative to the groups treated with L3D10+anti-PD-1 or control antibody(hIgG), which were fairly similar. The bone marrow from mice treatedwith 10D1+anti-PD-1 had a noticeably whiter color (FIG. 6A) and theisolated blood was almost completely white in color (FIG. 6B). Inaccordance with this, when differentiation of the red blood cells wasanalyzed using distribution of CD119 and CD71 markers, a statisticallysignificant reduction in the number of cells undergoing Stage IVdevelopment was observed in the 10D1+anti-PD-1 treated mice.Representative FACS profiles are shown in FIG. 6C, while summary dataare presented in FIG. 6D.

FIG. 7. Toxicity scores of mice treated with chimeric L3D10 and 10D1 incombination with anti-PD-1. This tissue data is summarized and shows thehigh toxicity scores of mice treated with 10D1+anti-PD-1 relative toL3D10+anti-PD-1 which has scores only marginally higher than the hIgGcontrol mouse group.

FIG. 8. L3D10 and 10D1 display similar binding patterns for plateimmobilized CTLA-4. ELISA plates were coated with 1 μg/ml of CTLA-4-Hisprotein (Sino Biological, China). The given concentration ofbiotinylated binding proteins were added and binding measured usingHRP-conjugated streptavidin. 10D1-1 and −2 are two independent materiallots of the same antibody. hIgG-Fc is a human Ig negative control.

FIG. 9. L3D10 displays reduced binding soluble CTLA-4. Givenconcentration of anti-human CTLA-4 mAbs were coated on the plateovernight, after washing and blocking with bovine serum albumin,biotinylated CTLA-4-Fc was added at 0.25 μg/ml. After incubation andwashing, the amounts of captured CTLA-4-Fc were measured usingHRP-labeled streptavidin.

FIG. 10. 10D1+anti-PD-1 do not have significant toxicity in theCtla-4^(h/m) mice as evidenced by normal body weight gains in mice thatreceived antibody treatment during the perinatal period. The micereceived treatments with given antibody or combinations on days 10, 13,16, 19 and 22 intraperitoneally (100 μg/mice/injection/antibody). Micewere weighed at least once every 3 days.

FIGS. 11A-B. Anti-tumor activity of humanized L3D10 antibodies comparedto 10D1. Using the MC38 mouse tumor model in human CTLA-4 knockin mice,the anti-tumor activity of humanized L3D10 antibodies was investigatedcompared to the chimeric L3D10 antibody and 10D1. FIG. 11A shows thetreatment schedule of the in vivo experiment. Mice were given a total of4 doses of antibody every 3 days starting on day 7 after inoculation.All humanized antibodies (n=6 per group) completely eradicated thetumors and were comparable to 10D1 (FIG. 11B).

FIG. 12. Comparison among 10D1, mAb4 and mAb5 females for their combinedtoxicity with anti-PD-1 mAb. Female Ctla-4^(h/h) mice were treated ondays 10 or 11 days after birth with four injections of antibodies (100μg/mice/injection, once every three days) or control Fc as specified inthe legends. Mice were weighted once every 3 days. Data shown are meansand SEM of % weight gain over a 30 day period. All mice were sacrificedon day 43 for histological analysis. The number of mice used per groupis shown in the parentheses of labels.

The p-values for comparisons between various treatments are as follows.

hIg vs. αPD1 + L3D10 P value = 0.16 hIg vs. αPD1 + hIg P value = 0.0384*hIg vs. αPD1 + 10D1 P value = <2e−16*** hIg vs. αPD1 + mAb4 P value =0.16 hIg vs. αPD1 + mAb5 P value = 0.00207** αPD1 + L3D10 vs. αPD1 + hIgP value = 0.00654** αPD1 + L3D10 vs. αPD1 + 10D1 P value = <2e−16***αPD1 + L3D10 vs. αPD1 + mAb4 P value = 0.492 αPD1 + L3D10 vs. αPD1 +mAb5 P value = 0.000124*** αPD1 + hIg vs. αPD1 + 10D1 P value =<2e−16*** αPD1 + hIg vs. αPD1 + mAb4 P value = 0.0579 αPD1 + hIg vs.αPD1 + mAb5 P value = 0.409 αPD1 + 10D1 vs. αPD1 + mAb4 P value =<2e−16*** αPD1 + 10D1 vs. αPD1 + mAb5 P value = <2e−16*** αPD1 + mAb4vs. αPD1 + mAb5 P value = 0.000446***

FIG. 13. Humanization of L3D10 does not affect binding to immobilizedCTLA-4. The capacity of the humanized L3D10 antibodies to bindimmobilized CTLA-4 was determined as described in FIG. 23. X-axisindicates the concentration of anti-CTLA-4 mAbs added into solution.Humanization does not affect binding to immobilized CTLA-4 and all threehumanized antibodies demonstrated similar binding to the parentalchimeric L3D10 antibody and 10D1. Similar patterns were observed whenCTLA-4-Ig was used instead of CTLA-4-his.

FIG. 14. Humanization further reduces L3D10 binding to soluble CTLA-4.The capacity of the humanized L3D10 antibodies to bind soluble CTLA-4was determined as described in FIG. 23. X-axis indicates theconcentration of anti-CTLA-4 mAbs coated onto ELISA plates. Humanizationfurther reduces binding to soluble CTLA-4 relative to the parental L3D10chimeric antibody. Similar patterns were observed when CTLA-4-Ig wasused instead of CTLA-4-his.

FIG. 15. Alignment of the human, macaque and mouse CTLA-4 extracellulardomains. The amino acid sequences of the human (Hm; amino acids 1-124 ofSEQ ID NO: 2), macaque (Mk) and mouse (Ms) CTLA-4 protein extracellulardomains are aligned and the conserved amino acids (relative to the humansequence) are shown with dashes (-). In order to help the alignment, themouse sequence has a deletion and insertion (relative to the human andmonkey sequences) at the positions highlighted. The known B7-1Igbinding; site is shown in bold and underlined. The sequences demonstratethat the human and monkey sequences are highly conserved, whereas themouse sequence has a number of amino acid differences. Based on thissequence alignment, 11 mutant (M1-M11) human CTLA-4Fc proteins weredesigned that incorporate murine specific amino acids—the amino acidsincorporated into each mutant protein are shown in blue.

FIGS. 16A and B. Amino acid sequence composition of the WT (SEQ ID NO:2) and mutant CTLA-4Fc proteins (SEQ ID NOs: 7-17). DNA constructsencoding the WT CTLA-4Fc protein and 11 mutant proteins, M1-M11,incorporating murine Ctla-4 amino acids were designed as shown. Theamino acid sequences are for mature proteins, including the IgG1 Fcportion, but not the signal peptide. The known B7-1Ig binding site isshown in large letters and double-underlined. The replaced murine aminoacid residues in the mutant are shown lower case. The IgG1 Fc portion ofthe proteins in underlined.

FIGS. 17A-D. Mutation in M11 (AA103-106, YLGI>fcGm) selectively abolishantibody binding to human CTLA-4. Data shown are means of duplicates,depicting the binding of B7-1Fc (FIG. 17A), L3D10 (FIG. 17B), mAb4 (FIG.17C), and mAb5 (FIG. 17D) binding to plate-coated hCTLA-4-Fc (opencircles), mCTLA-4-Fc (filled triangles), M11 (filled circles) andIgG1-Fc (open triangles).

FIG. 18. Mapping L3D10, mAb4 and mAb5 to an epitope adjacent to the B7-1binding site in a 3-D structure of the B7-1-CTLA-4 complex. The B7-1binding motif and antibody epitope are shaded differently. B7-1 isdepicted above CTLA-4 with a space-filled ribbon, while that of CTLA-4is depicted as an unfilled ribbon.

FIG. 19. Amino acid sequence composition of the mutant CTLA-4Fcproteins, M12-M17 (SEQ ID NOs: 18-23). DNA constructs encoding the 6mutant CTLA-4Fc proteins, M12-M17, incorporating murine Ctla-4 aminoacids were designed as shown. The amino acid sequences are for matureproteins, including the IgG1 Fc portion, but not the signal peptide. Theknown B7-1Ig binding site is shown in large letters anddouble-underlined. The replaced murine amino acid residues in the mutantare shown lower case. The IgG1 Fc portion of the proteins is underlined.

FIGS. 20A and B. Identifying CTLA-4Fc mutant proteins that retainbinding to biotinylated B7-1Fc, CTLA4-Fc fusion proteins M1-M17 werecoated onto 96-well plates. Biotinylated B7-1Fc at the givenconcentration was incubated with the plates and detected with.streptavidin-conjugated HRP. Data shown are means of duplicateexperiments.

FIGS. 21A and B. Identifying CTLA-4Fc mutant proteins that retainbinding to biotinylated B7-1 and B7-2 proteins. Mutant CTLA4-Fc fusionproteins (1 μg/ml) were coated onto 96-well plates. Biotinylated B7-1Fc(FIG. 21A) and B7-2Fc (FIG. 21B) were incubated. with the plates at thegiven concentrations and detected with streptavidin-conjugated HRP. Datashown are means of duplicate experiments.

FIGS. 22A and B. Identifying CTLA-4Fc mutant proteins that have lostbinding to biotinylated Ipilimumab (FIG. 22A) and Tremelimumab (FIG.22B). Mutant CTLA4-Fc fusion proteins (1 μg/ml) were coated onto 96-wellplates. Biotinylated antibody mAb2 was incubated with the plates at thegiven concentrations and detected with streptavidin-conjugated HRP. Datashown are means of duplicate experiments.

FIGS. 23A and B. Identifying CTLA-4Fc mutant proteins that have lostbinding to biotinylated mAb4. CTLA4-Fc fusion proteins M1-M17 werecoated onto 96-well plates. Biotinylated antibody mAb4 was incubatedwith the plates at the given concentrations and detected withstreptavidin-conjugated HRP. Data shown are means of duplicateexperiments.

FIGS. 24A and B. Identifying CTLA-4Fc mutant proteins that have lostbinding to biotinylated mAb5. CTLA4-Fc fusion proteins M1-M17 werecoated onto 96-well plates. Biotinylated antibody mAb5 was incubatedwith the plates at the given concentrations and detected withstreptavidin-conjugated HRP. Data shown are means of duplicateexperiments.

FIGS. 25A and B. Identifying CTLA-4Fc mutant proteins that have lostbinding to biotinylated mAb4 (FIG. 25A) and mAb5 (FIG. 25B). MutantCTLA4-Fc fusion proteins (1 μg/ml) were coated onto 96-well plates.Biotinylated antibody mAb4 (FIG. 25A) or mAb5 (FIG. 25B) was incubated.with the plates at the given concentrations and detected withstreptavidin-conjugated HRP. Data shown are means of duplicateexperiments.

FIGS. 26A-F. The therapeutic effect of Ipilumumab is not achieved byblocking B7-1 and/or B7-2. FIG. 26A. Confirmation of the blockingactivities of anti-B7 mAbs. CHO cells expressing mouse B7-1 or B7-2 wereincubated with a mixture of 20 mg/ml anti-B7-1 (upper panel) oranti-B7-2 (lower panel) and biotinylated human CTLA-4Fc (2 mg/ml) for 1hour. Alter washing away unbound proteins, the cell surface CTLA-4Fc wasdetected by PE-conjugated streptavidin and measured by flow cytometry.Data shown are representative FACS profiles and have been repeatedtwice. FIG. 26B. Diagram of the experimental design: MC38 tumor-bearingCtla-4^(h/m) mice received anti-B7-1 and anti-B7-2 antibodies (300μg/mouse/injection, once every 3 days for a total of 3 injections) incombination with either control Ig or Ipilimumab. Mice that receivedIpilimumab without anti-B7-1 and anti-B7-2 were used as a positivecontrol for tumor rejection and big was used as a negative control.FIGS. 26C and D. Saturation of B7-1 and B7-2 by antibody treatments asdiagrammed in FIG. 26B. The PBL from mice treated as shown in FIG. 26B,were stained with fluorochrome-conjugated anti-B7-1 and anti-B7-2 mAbsat 24 hours after anti-B7 treatment. PBL from Cd80^(−/−)Cd86^(−/−) micewere used as negative control. FIG. 26E. Functional blockade of B7 byanti-B7-1 and anti-B7-2 mAbs based on ablation of antibody responses.Sera were collected at day 22 after tumor challenge to evaluateanti-human IgG antibody responses. FIG. 26F. Saturating blocking of B7-1and B7-2 by anti-B7-1 and anti-B7-2 mAbs does not affect theimmunotherapeutic effect of Ipilimumab. Data shown are tumor volumesover time and have been repeated twice with similar results.

FIG. 27. CTLA-4-Fc fusion proteins M11 and M15 protect mice against irAEcaused by combination therapy with anti-PD-1 and anti-CTLA-4(Ipilimumab).

FIGS. 28A and B. CTLA4-Fc mutants M15 and M17 do not interfere with theimmunotherapeutic effect of Ipilimumab. The. Ctla-4^(h/m) heterozygousmice were transplanted with MC38 tumors and treated with Ipilimumab incombination with either control IgG Fc or CTLA-4 mutant proteins at timepoints indicated by the arrows. FIG. 28A. Reduction in tumor sizes. FIG.28B. Survival of tumor-bearing mice, using tumor sizes of reached 2 cmin diameter as early removal criteria.

FIGS. 29A and B. Protective function of M15 and M17-2 againstIpilimumab-induced weight-loss (FIG. 29A) and potential liver toxicityof M15 (FIG. 29B) in NSG mice reconstituted with human CD34⁺hematopoietic stem cells. 16 female humanized mice were randomized. into4 groups based on the weight human leukocyte reconstitution and % ofhuman T cells in the blood. The 4 groups were treated, respectively,with either control human IgG Fc (400 μg/injection), Ipilimumab (100μg/injection)+human IgG Fc (300 μg/injection), Ipilimumab (100μg/injection)+M15 (300 μg/injection), or Ipilimumab (100μg/injection)+M17-2 (300 μg/injection) on days 0, 3 and 6. On day 18,the mice received one more injection in which the dose of Ipilimumabwere 200 μg/mice+human IgG Fc (300 μg/injection), Ipilimumab (100μg/injection)+M15 (300 μg/injection), or Ipilimumab (100μg/injection)+M17-2 (300 μg/injection). Mice were monitored over 30 daysfor weight changes, using day 0 as 100%. FIG. 29B. Elevation of ALT wasobserved in mice that received Ipilimumab along with M15 but not M17-2.

FIGS. 30A-F. Differential effect of M15 and M17-2 in Ipilimumab-inducedT cell activation based on the analysis of spleen T cells on day 31.Mice were treated as described in FIG. 29 and sacrificed on day 31 forflow cytometry. CD4 (FIGS. 30A-C) and CD8 (FIGS. 30D-F) frequency of CD4and CD8 T cells (FIGS. 30A and D), central memory (CD40^(hi)CD62L^(hi))(FIGS. 30B and E) and effector memory cells (CD44^(hi)CD62L^(lo)) (FIGS.30C and F) cells.

FIG. 31. Differential effect of M15 and M17 on the expansion of NKTcells. As detailed in FIG. 30, except that NK T cells (CD3⁺CD56⁺) wereanalyzed. Mice were treated as described in FIG. 29 and sacrificed onday 31 for flow cytometry.

FIGS. 32A-D. Differential effect of M15 and M17-2 on the frequency ofTreg (FIGS. 32A and B) and their expression of CTLA-4 protein (FIGS. 32Cand D). Mice were treated as described in FIG. 29 and sacrificed on day31 for flow cytometry.

DETAILED DESCRIPTION

The inventors have discovered soluble CTLA-4 fusion proteins containingmutations that prevent their binding by most anti-CTLA-4 mAbs whileretaining their ability to bind B7-1 and B7-2 on antigen presentingcells (APC). Surprisingly, these fusion proteins prevent the autoimmuneadverse effects of anti-CTLA-4 mAbs without affecting the cancertherapeutic effects of anti-CTLA-4.

1. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6,6, 6.7, 6.8, 6,9 and 7.0 are explicitlycontemplated,

As used herein, the term “protein”, “peptide” or “polypeptide” refers toa chain of amino acids of any length, regardless of modification (e.g.,phosphorylation or glycosylation). The ammo acids may be natural,synthetic, or a modification or combination of natural and synthetic. Apolypeptide of the present invention may be a recombinant polypeptide, anatural polypeptide or a synthetic polypeptide, preferably a recombinantpolypeptide.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide. A“fragment” of a polypeptide thus refers to any subset of the polypeptidethat is a shorter polypeptide of the full length protein. Generally,fragments will be five or more amino acids in length.

As used herein, the term “soluble portion” of a protein means thatportion of the full length polypeptide that does not include any part ofthe transmembrane portion or segment. For example, with respect toCTLA-4, a soluble portion would include the extracellular portion (withor without the N-terminal signal sequence) but would not include anypart of the transmembrane portion (or, at least, not enough to reducesolubility). Thus, the ECD of human CTLA-4 is shown SEQ ID NO: 3 (i.e.,amino acids 36-161 of the full length sequence (SEQ ID NO: 1), whereamino acids 1-35 comprise the signal sequence and are not included inthe mature extracellular, and thus soluble, protein.

As used herein, the term “fusion protein” is defined as one or moreamino acid sequences joined together using methods known in the art. Thejoined amino acid sequences thereby form one fusion protein. Fusionproteins known in the art include the hexa-histidine peptide, thehemagglutinin “HA” tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson, I. A. et al. (1984) “TheStructure Of An Antigenic Determinant In A Protein,” Cell, 37:767-778)and the “flag” tag (Knappik, A. et al. (1994) “An improved Affinity TagBased On The FLAG Peptide For The Detection And Purification OfRecombinant Antibody Fragments,” Biotechniques 17(4):754-761).

A derivative, analog or homolog, of a polypeptide. (or fragment thereof)of the invention may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (tor example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchderivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

As used herein, the term “antibody” is intended to denote animmunoglobulin molecule that possesses a “variable region” antigenrecognition site. The term “variable region” is intended to distinguishsuch domain of the immunoglobulin from domains that are broadly sharedby antibodies (such as an antibody Fc domain). The variable regioncomprises a “hypervariable region” whose residues are responsible forantigen binding. The hypervariable region comprises amino acid residuesfrom a “Complementarity Determining Region” or “CDR” (i.e., typically atapproximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in thelight chain variable domain and at approximately residues 27-35 (H1),50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; ref. 44)and/or those residues from a “hypervariable loop” (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Ref. 45). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined. The term antibody includes monoclonal antibodies,multi-specific antibodies, human antibodies, humanized antibodies,synthetic antibodies, chimeric antibodies, camelized antibodies, singlechain antibodies, disulfide-linked Fvs (sdFv), intrabodies, andanti-idiotypic (anti-Id) antibodies (including, e.g. anti-Id andanti-anti-Id antibodies to antibodies of the invention). In particular,such antibodies include immunoglobulin molecules of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the term “active fragment” refers to a portion of anatural polypeptide or antibody, or a polypeptide with high sequencehomology (for example, at least 60%, 85%, 90%, 95%, 98%, or 99% aminoacid sequence identity) to a natural polypeptide or antibody and whichretains biological activity. Representative examples of “biologicalactivity” include the ability to bind B7 proteins and to bind theirnatural receptors or to be bound by a specific antibody. For example, anactive fragment of CTLA-4 would be capable of binding B7.1 or B7.2 or bybinding to a ligand of CTLA-4. In preferred embodiments, such a fragmentwould consist of the extracellular domain (ECD) of a CTLA-4 protein.

As used herein the term “isolated” is meant to describe a compound ofinterest (e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurse.g. separated from its natural milieu such as by concentrating apeptide to a concentration at which it is not found in nature.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 amino acids.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

As used herein, a “variant” polypeptide contains at least one ammo acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide. As used herein, an “amino acidsequence alteration” can be, for example, a substitution, which may beconservative, a deletion, or an insertion of one or more amino acids.Variant may also mean a protein with an amino acid sequence that issubstantially identical to a referenced protein with an amino acidsequence, and may retain at least one biological activity. A variant maybe a derivative, analog or homolog, of a polypeptide. A variant may alsobe a soluble portion of polypeptide. A conservative substitution of anamino acid, i.e., replacing an amino acid with a different amino acid ofsimilar properties hydrophilicity, degree and distribution of chargedregions) is recognized in the art as typically involving a minor change.These minor changes can be identified, in part, by considering thehydropathic index of amino acids, as understood in the art. Kyte et al.,J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids having;hydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. B7-CTLA-4 Interactions Anti Immunotherapy

The ability of T cells to optimally mediate an immune response againstan antigen requires two distinct signaling interactions. First, antigenthat has been arrayed on the surface of antigen-presenting cells (APC)must be presented to antigen-specific naive T cells in the form of MHC:peptide complex (1, 2). Such presentation delivers a signal via the Tcell receptor (TCR) that directs the T cell to initiate an immuneresponse that will be specific to the presented antigen. Second, aseries of co-stimulatory signals, mediated through interactions betweenthe APC and distinct T cell surface molecules, triggers first theactivation and proliferation of the T cells and ultimately theirinhibition (3-5). Thus, the first signal confers specificity to theimmune response whereas the second signal serves to determine thenature, magnitude and duration of the response while limiting immunityto self. Of particular importance among, these second signal moleculesis binding between the B7.1 (CD80) (6) and B7.2 (CD86) (7-9) ligands ofthe APC and the CD28 and CTLA-4 receptors (10-12) of the T-lymphocyte.

CD28 (Cluster of Differentiation 28) and CytotoxicT-lymphocyte-associated antigen-4 (CTLA-4) are members of theimmunoglobulin superfamily of proteins and are involved in theregulation of T-cell activation. CD28 is constitutively expressed onnaive T cells and binds B7.1 and B7.2 providing a co-stimulatory signalrequired for T cell activation and survival. In contrast, upon cellactivation, CTLA-4 is upregulated on I cells and competes with CD28 forbinding to B7.1 and B7.2, thereby transmitting a suppressive signal forT cell activation.

B7 family molecules have a membrane proximal IgC (constant) domain and amembrane distal IgV (variable) domain. The CD28-like family of receptorsfor these ligands share a common extracellular IgV-tike domain.Interactions of receptor-ligand pairs are mediated predominantly throughresidues in the IgV domains of the ligands and receptors (Schwartz, etal., Nature Immunol., 3:427-434 (2002)). Crystallographic analysisrevealed that the CTLA-4/B7 binding interface is dominated by theinteraction of the CDR3-analogous loop from CTLA-4, composed of a MYPPPYmotif (Schwartz, et al., Nature, 410:604-608 (2001); and Stamper, etal., Nature. 410:608-611 (2001)).

Antibodies against CTLA-4 have been shown to block the interactionbetween CTLA-4 and the costimulatory molecules B7.1 and B7.2 in vitroThis blockade removes the CTLA-4-mediated inhibitory signal on T-cellsand thereby stimulates a natural immune response that can be used as atherapy against cancer. Treatment with anti-CTLA-4 antibodies has beenshown to be a powerful tool for enhancing anti-tumor immunity inpreclinical models (10). Monotherapy with an antibody against CTLA-4promoted rejection of transplantable tumors of various origins. Based onpromising preclinical tumor model studies, the clinical potential ofantibodies against CTLA-4 has been explored m different humanmalignancies. Although anti-CTLA-4 (Ipilimumab, marketed as Yervoy,disclosed in U.S. Pat. No. 6,984,720) has demonstrated efficacy intreating melanoma, treatment and targeting of CTLA-4 is associated withautoimmune like toxicities. Characteristic side effects from inhibitionof CTLA-4 are generally called immune-related adverse events (irAEs) andthe most common irAEs are skin rash, hepatitis, colitis andendocrinopathies, particularly hypopituitarism. Therefore, there is adesire to improve the safety of anti-CTLA-4 antibodies by reducing theassociated irAEs.

The inventors have surprisingly discovered that both clinically proventherapeutic anti-human CTLA-4 mAb and two anti-mouse Ctla-4 mAbs inducetumor rejection without blocking B7-CTLA-4 interactions underphysiologically relevant conditions. Therefore, blocking CTLA-4interactions with B7.1 or B7.2 is not necessary for tumor rejection evenfor the mAb that can potently block these B7-CTLA-4 interactions. Thesedata refute the hypothesis that anti-CTLA-4 mAb confersimmunotherapeutic effect through checkpoint blockade (108). Furthermore,the inventors have identified an anti-CTLA-4 antibody, L3D10, withreduced immune related toxicities demonstrating that cancer immunity andirAEs can be uncoupled genetically.

3. Soluble CTLA-4

Accumulating data demonstrated that the human CTLA-4 gene encodes twodifferent isoforms of proteins through alternative splicing: one with atrans-membrane domain which is thus likely to be anchored in themembrane, and another that lacks the trans-membrane domain and ispredicted to be secreted (sCTLA-4, SEC) ID NO: 4) (128). Importantly,genetic studies demonstrated that a polymorphism of CTLA-4 that reducesthe relative abundance of the soluble isoform strongly associates withmultiple autoimmune diseases (64). The fact that subjects withautoimmune prone alleles express less sCTLA-4 mRNA suggests that sCTLA-4may be protective. This is notion is supported by the broad therapeuticeffect of abatacept (129,130), which is a form of soluble CTLA-4, and bythe genetic study in which the selective ablation of the sCTLA-4 isoformaccelerated the development of type is diabetes in the mice (131).

Based on these genetic data it the inventors had the insight that theantibody that shows the poorest binding to sCTLA-4 should be associatedwith the fewest irAE. Indeed, the inventors identified an anti-CTLA-4antibody, L3D10, with reduced immune related toxicities that was shownto have reduced binding to sCTLA-4 relative to membrane bound orimmobilized CTLA-4. Based on the impact of the antibodies on the bodyweight gain in mice treated with anti-CTLA-4 antibodies during theperinatal period, a strong correlation was found among four anti-CTLA-4mAbs: Ipilimumab has the strongest binding for sCTLA-4 and is the mosttoxic anti-CTLA-4, whereas L3D10 had the weakest binding to sCTLA-4 andwas the least toxic. Furthermore, humanization of the L3D10 antibodypreferentially reduced binding to sCTLA-4 and appeared to furtherimprove the safety profile over the parent antibody.

In order to map the CTLA-4 binding epitope of the L3D10 parent antibodyand humanized variants, the inventors took advantage of the fact thatthe mouse and human CTLA-4 proteins are cross-reactive between speciesto B7-1, but not to the anti-CTLA-4 antibodies. Accordingly, theinventors designed a number of mutants of the human CTLA-4Fc protein inwhich clusters of amino acids from the human CTLA-4 protein werereplaced with amino acids from the murine Ctla-4 protein (SEQ ID NO: 5).As the anti-CTLA-4 antibodies used in this study do not bind to murineCtla-4, binding of the anti-human CTLA-4 antibodies should be abolishedwhen key residues of the antibody binding epitope are replaced withmurine amino acids. Accordingly, the inventors have shown that the L3D10binding site of CTLA-4 maps directly adjacent to the B7-1 binding motif,MYPPPY (SEQ ID NO: 50). This correlates well with the demonstratedability of the antibody to block B7-CTLA-4 interactions both in vitroand in vivo. By contrast, Ipilimumab, does not block binding of B7.1 orB7.2 to abatacept under physiological conditions and so it must alsobind a region that does not include the MYPPPY motif. However, as itdoes not show reducing binding to sCTLA-4, the binding domain must bedifferent from antibody L3D10.

Endogenous sCTLA-4 is produced by fusion of the truncated C-terminal endof the extracellular IgV domain directly to the intracellular domain,without the intervening transmembrane domain. Accordingly, there areonly 12 amino acids of the CTLA-4 extracellular domain C-terminal to theMYPPPY motif in sCTLA-4, whereas the endogenous membrane hound versionhas 21 amino acids. Based on this, without being hound by theory, itappears that an antibody that binds to polymorphic C-terminal domainresidues, such as L3D10, is more likely to lose reactivity to sCTLA-4.Furthermore, the inventors have demonstrated that it is possible tomutate these amino acids in the CTLA-4Fc protein in this region so thatthey no longer bind anti-CTLA-4 antibodies.

4. Recombinant CTLA-4 Proteins

CTLA-4Fc fusion protein (abatacept, marketed as Orencia) is a selectivecostimulation modulator comprising the extracellular domain of CTLA-4fused to the Fc region of human IgG1 (shown in FIG. 1B; SEQ ID NO:2)that inhibits T cell (T lymphocyte) activation by binding to B7.1 andB7.2, thereby blocking interaction with CD28. In vitro, abataceptdecreases T cell proliferation and inhibits the production of thecytokines TNF alpha (TNFα), interferon-γ, and interleukin-2. Activated Tlymphocytes are implicated in the pathogenesis of Rheumatoid Arthritis(RA) and are found in the synovium of patients with RA. In a ratcollagen-induced arthritis model, abatacept suppresses inflammation,decreases anti-collagen antibody production, and reduces antigenspecific production of interferon-γ. Based on this preclinical activity,abatacept has been developed and approved for the treatment of RA andJuvenile Idiopathic Arthritis (JIA) in humans.

Although capable of reducing immune responses, CTLA-4Fc fusion proteinscomprising the endogenous CTLA-4 extracellular domain, such asabatacept, are incapable of reducing or mitigating the immune relatedtoxicities associated with anti-CTLA-4 immunotherapies as the activityof the two molecules are offsetting. The CTLA-4 fusion protein such asthe abatacept will not only reduce the therapeutic effect of anti-CTLA-4mAbs by binding directly to the antibodies to prevent them from bindingto endogenous cell-associated CTLA-4 molecules, but will also berendered ineffective themselves as they will be cleared from circulationafter forming immune-complex with circulating anti-CTLA-4 mAbs.

Given that both blocking and non-blocking anti-CTLA-4 antibodies haveanti-tumor effects and antibodies demonstrating reduced irAEs areassociated with reduced binding to sCTLA-4, the inventors engineered apanel of soluble CTLA-4 proteins with mutations that prevent binding bymost anti-CTLA-4 mAbs that were tested while retaining their ability tobind B7-1 and B7-2 on the antigen-presenting cells, which is criticalfor the pathogenesis of autoimmune disease and irAEs. Such proteins, byvirtue of their retained B7 binding activity, can be used in thetreatment. of autoimmune diseases such as RA and JIA. Furthermore, suchproteins can be used in combination with anti-CTLA-4 immunotherapytreatment to reduce the associated irAEs, while leaving the anti-tumoractivity of the immunotherapy intact.

a. CTLA-4 Proteins

Provided herein is a CTLA-4 protein comprising (a) the extracellulardomain of mature human CTLA-4 (SEQ ID NO: 3) or mouse CTLA-4 (SEQ ID NO:6), (b) an amino acid set forth in one of SEQ ID NOS: 24-40 and 46-49,(c) a variant of the foregoing, or (d) an active fragment of theforegoing. The CTLA-4 protein may be soluble. The CTLA-4 protein mayfurther comprise an N-terminal signal peptide. The CTLA-4 protein mayretain the ability to bind human B7.1 and B7.2 but lack the ability tobind an anti-CTLA-4 antibody. In one specific embodiment, the CTLA-4protein does not bind to Ipilimumab or Tremelimumab under physiologicalconditions. In particular, the CTLA-4 protein may comprise an amino acidsequence set forth in SEQ ID NO: 34, 36, 38, 39, 40, 46, 47, or 48. TheCTLA-4 protein may be isolated.

b. CTLA-4 Fusion Proteins

The CTLA-4 protein may be fused at its N- or C-terminal end to anotherprotein. The other protein may be a portion of a mammalian Ig protein,which may be human or mouse. The portion may comprise a Fc region of theIQ protein. The Fc region may comprise the hinge region and CH2 and CH3domains of the Ig protein. The Ig protein may be human IgG1, IgG2, IgG3,IgG4, or IgA. The Ig protein may also be IgM, and the Fc portion maycomprise the hinge region and CH3 and CH4 domains of IgM. In oneembodiment, the Fc region is human. IgG1 comprising the amino acidsequence set forth in SEQ ID NO: 41. The CTLA-4 protein may comprise anamino acid sequence selected from the group consisting of SEQ ID NOS:7-23 and 42-45, and particularly selected from the group consisting ofSEQ ID NO: 17, 19, 21, 22, 23, 42, 43, and 44.

The CTLA-4 protein may also be fused at its N- or C-terminus to aprotein tag, which may comprise GST, His, or FLAG. Methods for makingfusion proteins and purifying fusion proteins are well known in the art.

5. Anti-B7 Antibodies

Another embodiment of the invention relates to an anti-B7 antibodycomposition that binds at least one of human B7-1 and B7-2. The anti-B7antibody may bind to one or both of endogenous B7-1 and B7-2, and mayneutralize activity without affecting the cancer immunotherapeuticactivity of anti-CTLA-4 antibodies. The anti-B7 antibody may behumanized to minimize anti-drug antibodies. The anti-B7 antibody may bea monoclonal antibody, and may be cross-reactive with both B7-1 andB7-2. In yet another embodiment, a bispecific antibody may compriseantigen-binding domains of two antibodies, respectively binding to B7-1or B7-2

6. Production

The CTLA-4 protein or anti-B7 antibody may be prepared using aeukaryotic expression system. The expression system may entailexpression from a vector in mammalian cells, such as Chinese HamsterOvary (CHO) cells. The system may also be a viral vector, such as areplication-defective retroviral vector that may be used to infecteukaryotic cells. The CTLA-4 protein may also be produced from a stablecell line that expresses CTLA-4 protein from a vector or a portion of avector that has been integrated into the cellular genome. The stablecell line may express CTLA-4 protein from an integratedreplication-defective retroviral vector. The expression system may beGPEx™.

The CTLA-4 protein or anti-B7 antibody can be purified using, forexample, chromatographic methods such as affinity chromatography, ionexhange chromatography, hydrophobic interaction chromatography, DEAE ionexchange, gel filtration, and hydroxylapatite chromatography. In someembodiments, fusion proteins can be engineered to contain an additionaldomain containing amino acid sequence that allows the polypeptides to becaptured onto an affinity matrix. For example, a CTLA-4 protein oranti-B7 antibody comprising the Fc region of an immunoglobulin domaincan be isolated from cell culture supernatant or a cytoplasmic extractusing a protein A column. In addition, a tag such as c-myc,hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aidpolypeptide purification. Such tags can be inserted anywhere within thepolypeptide, including at either the carboxyl or amino terminus. Otherfusions that can be useful include enzymes that aid in the detection ofthe polypeptide, such as alkaline phosphatase. Immunoaffinitychromatography also can be used to purify polypeptides. Fusion proteinscan additionally be engineered to contain a secretory signal (if thereis not a secretory signal already present) that causes the fusionprotein to be secreted by the cells in which it is produced. Thesecreted fusion proteins can then conveniently be isolated from the cellmedia.

7. Pharmaceutical Compositions

The invention further concerns a pharmaceutical composition comprising atherapeutically effective amount of one or more of the above-describedCTLA-4 proteins and anti-B7 antibodies, and a physiologically acceptablecarrier or excipient. Preferably, compositions of the invention comprisea prophylactically or therapeutically effective amount of the CTLA-4protein or anti-B7 antibody and a pharmaceutically acceptable carrier

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers may be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, may also contain manor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions may take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention may besupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline may be provided so that the ingredients may bemixed prior to administration.

The compositions of the invention may be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limitedto, those formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

8. Methods of Treatment a. Autoimmune Disease

The CTLA-4 protein or anti-B7 antibody, or pharmaceutical compositionthereof may be used to treat an inflammatory or autoimmune disease.Representative inflammatory or autoimmune diseases and disorders thatmay be treated using B7-H4 fusion polypeptides include, but are notlimited to, rheumatoid arthritis (RA), systemic lupus erythematosus(SLE), alopecia areata, anklosing spondylitis, antiphospholipidsyndrome, autoimmune Addison's disease, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome (alps), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, bullous pemphigoid, cardiomyopathy, celiacsprue-dermatitis, chronic fatigue syndrome immune deficiency, syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricialpemphigoid, cold agglutinin disease, Crest syndrome, Crohn's disease,Dego's disease, dermatomyositis, dermatomyositis-juvenile, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,grave's disease, guillain-barre, hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), Iganephropathy, insulin dependent diabetes (Type I), juvenile arthritis,Meniere's disease, mixed connective tissue disease, multiple sclerosis(MS), myasthenia gravis, pemphigus vulgaris, pernicious anemia,polyarteritis nodosa, polychondritis, polyglancular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis. Raynaud'sphenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis,scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis,temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis,vasculitis, vitiligo. and Wegener's granulomatosis. In a particularembodiment, the autoimmune disease may be rheumatoid arthritis (RA) orJuvenile Idiopathic Arthritis (JIA). The CTLA-4 protein or anti-B7antibody, or pharmaceutical composition thereof may be administered to asubject in need thereof. The subject may be a mammal such as a. human.

The CTLA-4 protein or anti-B7 antibody, or pharmaceutical compositionthereof may be combined with another drug, such as a disease-modifyingantirheumatic drug (DMARD). The drug may be a nonsteriodanti-inflammatory drug (NSAID), which may be a propionic acidderivative, an acetic acid derivative, an enolic acid derivative, afenamic acid derivative, or a selective Cox2 inhibitor. The drug mayalso be a corticosteroid or Methotrexate. The drug may be a biologic,which may be a TNF-α antagonist such as an anti-TNF-α antibody or afusion protein that binds to TNF-α (Enbrel), an anti-CD20 mAb, anantagonist of costimulatory molecule CD80 and CD86 such as a monoclonalantibody or a fusion protein (CTLA-4Ig) that binds to the two molecules,or an antagonist for a receptor of either IL-1 or IL-6. The CTLA-4protein or anti-B7 antibody, or pharmaceutical composition thereof, andthe other drug may be administrated together or sequentially.

b. Immunotherapy

The CTLA-4 protein or anti-B7 antibody, or pharmaceutical compositionthereof may also be used to mitigate, reduce or treat the immune relatedadverse events (irAEs) associated with anti-CTLA-4 immunotherapy incancer patients. The CTLA-4 protein may be administered prophylactically(before the emergence of irAEs) or therapeutically (after the emergenceof irAEs). In particular, the CTLA-4 protein or anti-B7 antibody, orpharmaceutical composition thereof may be administered to a subject incombination with, or on a background of, anti-CTLA-4 immunotherapy. Thesubject may be cancer patient. In one embodiment, the CTLA-4 protein oranti-B7 antibody, or pharmaceutical composition thereof is usedprophylactically to prevent irAEs before anti-CTLA-4 treatment isinitiated or the before the clinical signs of irAEs emerge. In anotherembodiment, the CTLA-4 protein or anti-B7 antibody, or pharmaceuticalcomposition thereof is used therapeutically to treat irAEs afteranti-CTLA-4 treatment is initiated and the clinical symptoms arediagnosed.

c. Methods of Administration

The CTLA-4 protein or anti-B7 antibody, or pharmaceutical compositionthereof may be administered by a method including, but not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal and oral routes). In a specific embodiment, the CTLA-4protein or anti-B7 antibody, or pharmaceutical composition thereof isadministered intramuscularly, intravenously, or subcutaneously. Thecomposition may be administered by any convenient route, for example, byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local.

EXAMPLES Example 1 Chimeric L3D10 Antibody has Equivalent Activity as10D1 in Causing Tumor Rejection

In the clinic, the anti-CTLA-4 antibody, Ipilimumab (mab 10D1), has beenshown to improve the survival of cancer patients but induces significantautoimmune adverse effect. Using human CTLA-4 gene knock-in mice andhu-PBL-Scid mice, it was previously demonstrated that mouse anti-humanCTLA-4 antibodies reduced tumor growth, and identified L3D10 as the mosteffective among the panel of mAbs tested (21).

The availability of human CTLA-4 gene knockin mice (20) provided with anunprecedented opportunity to test biological activity of the chimericL3D10 anti-human CTLA-4 antibody, comprising the parental L3D10 variableregion and human IgG1 constant domain (the Fc region), with clinicallyused anti-CTLA-4 mAb, 10D1. In this humanized mouse model, a CTLA-4 geneencoding a product with 100% identity to human CTLA-4 protein isexpressed under the control of endogenous mouse CTLA-4 locus. When theanti-tumor activity of the chimeric L3D10 and 10D1 were directlycompared in the MC38 tumor model in human CTLA-4-knockin mice, it isclear that both antibodies were comparable in causing tumor rejection,whereas the tumors grew progressively in IgG control group. FIG. 2 showsthe results of antibody treatment on tumor size from duplicateexperiments.

Example 2 Reduction of Autoimmune Adverse Effect by OtherImmunotherapeutic Antibodies

Recent clinical studies have revealed that combination therapy betweenanti-PD-1 and anti-CTLA-4 mAb further increase the survival of end-stagemelanoma patients. However, 55% of the patients that received thecombination therapy developed grades 3 and 4 immune related adverseevents (irAEs). It is therefore critical to develop antibodies with lesstoxicity. We have developed an in vivo model that recapitulates theirAEs associated with the combination therapy of anti-CTLA-4 andanti-PD-1 mAbs observed in the clinic. In this model we treated humanCTLA-4 gene knockin mice (CTLA-4^(h/h)) during the perinatal period withhigh doses of anti-PD-1 and anti-CTLA-4 mAbs. We found that while theyoung mice tolerate treatment of individual mAbs, combination therapywith anti-PD-1 and 10D1 causes severe irAE with multiple organinflammation, anemia and, as shown in FIG. 3, severely stunted growth.in contrast, when combined with anti-PD-1, chimeric L3D10 exhibits onlymild irAE as demonstrated by normal weight gain.

To further examine to relative toxicity or chimeric L3D10 compared to10D1 when administered in combination with anti-PD-1, we looked at thepathalogical effects in the CTLA-4^(h/h) knockin mice at 42 days afteradministration. As shown in FIG. 4, terminal body weight (day 42) inmice treated with L3D10+anti-PD-1 was similar to mice treated with hIgGnegative control antibody. However, by comparison, the weight of micetreated with 10D1+anti-PD-1 was much lower. Accordingly, when we lookedat the gross anatomy of these mice, the Uterus/Ovary/Bladder and thymuswere noticably smaller in mice treated with 10D1+PD-1 (FIG. 5). Again,the organs in mice treated with L3D10+anti-PD-1 was comparable to hIgGcontrol. In contrast, the hearts dissected from mice treated with 10D1appeared slightly larger in size with a noticeably whiter appearance. Asa result we decided to look at erythropoiesis within the mice andobserved clear differences in the mice treated with 10D1+anti-PD-1relative to the groups treated with L3D10+anti-PD-1 or control antibody,which were fairly similar. As shown in FIG. 6A. the bone marrow frommice treated with 10D1+anti-PD-1 had a noticeably whiter color and theisolated blood was almost completely white in color (FIG. 6B). inaccordance with this, when we took at closer look at the cellsundergoing the different stages of blood development using CD71 and.CD119 markers. Representative FACS profiles are shown in FIG. 6C, whilesummary data are presented in FIG. 6d . These data revealed astatistically significant reduction in the number of cells undergoingStage IV development in the 10D1+anti-PD1 treated mice (FIG. 6D).

To further determine the toxicology of L3D10 vs 10D1 in combination withanti-PD-1, we performed histological analysis of the heart, lung,salivary gland and the kidney and liver following fixation in 10%formalin for at least 24 hours. In each of the tissues studied, micetreated with 10D1+anti-PD-1 displayed a high level of T cellinfiltration. The toxicity score, based on severity of inflammation, aresummarized in FIG. 7, which shows the high toxicity scores of micetreated with 10D1+anti-PD-1 relative to L3D10+anti-PD-1 which has scoresonly marginally higher than the bIgG control mouse group.

Example 3 L3D10 has reduced binding for soluble CTLA-4

L3D10 and 10D1 display similar binding patterns for plate immobilizedCTLA-4 (FIG. 8). As a possible explanation for the reduced toxicity ofL3D10 relative to 10D1, particularly the increased T cellinfiltration/activity associated with 10D1, we decided to look at thebinding to soluble CTLA-4. We chose to look at this because theassociation between CTLA-4 polymorphism and multiple autoimmune diseasesrelates to the defective production of soluble CTLA-4 (67) and geneticsilencing of the sCTLA-4 isoform increased the onset of type I diabetesin mice (131). Furthermore, soluble CTLA-4 (abatacept and belatacept) isa widely used drug for immune suppression. In accordance with this idea.when we looked at the relative binding to soluble CTLA-4, we observed amarked decrease in the binding of L3D10 relative to 10D1 (FIG. 9).

We have demonstrated that anti-CTLA-4 mAb induce robust tumor injectionin heterozygous Ctla-4^(h/m) mice in which only 50% of CTLA-4 moleculescan bind to anti-human CTLA-4 mAbs. To determine if engagement of 50% ofCTLA-4 is sufficient to induce irAE, we treated the Ctla-4^(h/m) micewith anti-PD-1+10D1. As shown in FIG. 10. anti-PD-1+10D1 failed toinduce weight loss in the Ctla-4^(h/m) mice. Therefore, irAE and cancerimmunity can be uncoupled genetically.

In vivo activity demonstrates that the L3D10 antibody retains itsanti-tumor activity but displays reduced autoimmune adverse effectobserved with other immunotherapeutic antibodies such as 10D1,indicating it is possible to enhance anti-tumor activity withoutexacerbating autoimmune adverse events. Accordingly, autoimmune sideeffects are not a necessary price for cancer immunity and that it ispossible to uncouple these two activities. Characterization of L3D10demonstrated that its ability to block the interaction of CTLA-4 withB7.1 and B7.2 is more effective than by 10D1. Further characterizationdemonstrates that L3D10 and 10D1 bind to immobilized CTLA-4 with asimilar binding profile. However, L3D10 demonstrates much lower bindingaffinity to soluble CTLA-4 than 10D1.

Example 4 Humanized L3D10 anti-CTLA-4 antibodies

Humanized L3D 10 antibodies were designed by creating multiple hybridsequences that fuse select parts of the parental antibody sequence withthe human framework sequences, including grafting of the CDR sequencesinto acceptor frameworks. We evaluated the anti-tumor activity of threeof the humanized antibodies (mAb4, mAb5 and mAb6) compared to 10D1 andthe chimeric L3D10 antibody using the syngeneic MC38 mouse tumor modelin human CTLA-4-knockin mice described in Example 1 above. FIG. 11Ashows the treatment schedule of the in vivo experiment; mice were givena total of 4 doses of antibody every 3 days starting on day 7 afterinoculation. As shown in FIG. 11B, all humanized antibodies completelyeradicated the tumor and were comparable to 10D1. Similar efficacy wasseen using the syngeneic MC38 mouse tumor model in heterozygousCtla-4^(h/m) mice and the syngeneic B16-F1 melanoma mouse tumor model inhuman CTLA-4-knockin mice (data not shown).

To test if the superior safety profiles of L3D10 can be maintained afterhumanization, we compared mAb4 and mAb5 with 10D1 for their adverseeffects when used in combination with anti-PD-1. As shown in FIG. 12,both mAb4 and mAb5 are less toxic than 10D1 when used in combinationwith anti-PD-1.

Example 5 Binding Characteristics of the Humanized Anti-CTLA-4Antibodies

In order to confirm that the humanized antibodies retained their CTLA-4binding characteristics, we looked at binding to immobilized and platebound CTLA-4. Humanization did not affect binding to immobilized CTLA-4and all 3 humanized antibodies demonstrated similar binding to theparental chimeric L3D10 antibody (FIG. 13). However, humanizationfurther reduces L3D10 binding to soluble CTLA-4 (FIG. 14).

We have demonstrated that chimeric L3D10 has a 1000-fold higher B7blocking activity than 10D1. This raised an interesting possibility thatblocking B7-CTLA-4 interactions may explain its lack of irAE. However,neither mAb4 nor mAb5 block B7-CTL-A4 interactions in vitro and in vivo.The fact that mAb4 and mAb5 show diminished irAE further supported thenotion that blocking B7-CTLA-4 interaction is not responsible forimproved safety of L3D10.

Given the proposed role for CTLA-4 in the protection against autoimmunediseases, we proposed reduced binding to soluble CTLA-4 as an underlyingmechanism for improved safety profiles. To test this hypothesis, we usedthe growth weight gain among the female mice that receivedanti-PD-1+anti-CTLA-4 mAbs during the perinatal period as the basicindicator for irAE. Severe reduction in weight gain was observed in themice that received both 10D1 and anti-PD-1, whereas those that receivedmAb5+anti-PD-1 had the lowest irAE, followed by mAb4 and then L3D10(data not shown). The strict inverse correlation with reduced binding tosCTLA-4 are consistent with the central hypothesis.

Example 6 Epitope Mapping of the L3D10 and Humanized Antibodies

In order to map the CTLA-4 binding epitope of the L3D10 antibody and thehumanized variants, mAb4 and mAb5, we took advantage of the fact thatthe mouse and human CTLA-4 proteins are cross-reactive to B7-1, but notto the anti-CTLA-4 antibodies. The fact that anti-human CTLA-4antibodies do not cross react with murine Ctla-4, presumably reflectsdifferences in the amino acid sequence between human and mouse CTLA-4 inthe extracellular domain. FIG. 15 shows the alignment of the human,macaque and mouse CTLA-4 extracellular domains and highlight thesequence conservation between human and macaque, while showing thenumerous differences between the murine and primate sequences. Due toconservation of the MYPPPY binding motif (SEQ ID NO: 50), mouse andhuman CTLA-4 proteins are cross-reactive to B7-1 (72).

Accordingly, we designed 11 mutants of the human CTLA-4Fc protein,designated M1-M11 (SEQ ID NOS: 7-17) in which clusters of amino acidsfrom the human CTLA-4 protein were replaced with amino acids from themurine Ctla-4 protein. The amino acids incorporated into each of the 11mutants is shown in FIG. 15, and the amino acids sequences of the WT andmutant CTLA-4Fc proteins is shown in FIGS. 16A and B. As the anti-CTLA-4antibodies used in this study do not bind to murine Ctla-4, binding ofthe anti-human CTLA-4 antibodies should be abolished when key residuesof the antibody binding epitope are replaced with murine amino acids.

DNA vectors encoding 11 CTLA-4Fc mutant proteins were constructed basedon the wild type human CTLA-4Fc sequence and proteins were produced bytransient transfection in HEK293 at the 0.01 mL scale followed byone-step Protein A chromatography purification, and the yield isprovided in Table 1. Many of the mutations appear to affect proteinexpression as indicated by their yields relative to the WT humanCTLA-4Fc protein.

TABLE 1 WT and mutant CTLA-4Fc proteins produced transiently in HEK293cells. Protein name Yield (mg) CTLA-4Fc WT control 0.72 Mutant 1 1.29Mutant 2 0.03 Mutant 3 0.21 Mutant 4 0.11 Mutant 5 1.89 Mutant 6 0.38Mutant 7 0.25 Mutant 8 1.61 Mutant 9 0.01 Mutant 10 0.04 Mutant 11 1.70

The capacity of chimeric L3D10 and the humanized antibodies mAb4 andmAb5 to bind the immobilized CTLA-4Fc mutant constructs was thendetermined by ELISA in which plates were coated with the CTLA-4 mutantproteins at 1 μg/mL and biotinylated anti-CTLA-4 antibodies, or B7-1 Igcontrol protein, were added and binding measured using HRP-conjugatedstreptavidin. The results of binding assays are shown in Tables 2-5. Asexpected, all 4 binding proteins demonstrated nice dose-dependentbinding for the WT CTLA-4Fc protein. However, mutations that wereintroduced into the M9 and M10 proteins appear to alter the overallstructure and these mutants failed to bind B7-1Fc. Mutations introducedin M2 and M4 also partially altered CTLA-4 conformation as indicated byreduced binding relative to the WT protein. Consistent with this notion,all 4 of these mutants (M2, M4, M9 and M10) were expressed at much loweryield (Table 1). in contrast, using binding to the WT CTLA-4Fc proteinand binding of the B7-1Fc proteins as references, M11 clearly stands outas a protein that is expressed well, binds B7-1Fc efficiently but failedto hind two humanized anti-CTLA-4 antibodies. Its binding to originalL3D10 is also reduced by approximately 100-fold (Table 3). As expected,the mutations that affect the overall conformation also affected thebinding to the anti-CTLA-4 antibodies.

TABLE 2 Integrity of CTLA-4Ig mutants as indicated by their binding toB7-1 Ig fusion protein. Binding to CTLA-4Fc proteins was performed byELISA, with the amounts of biotinylated protein bound measured byhorse-radish peroxidase (HRP)-conjugated streptavidin. Values shown arethe OD450 measurements. Protein Conc. WT M1 M2 M3 M4 M5 M6 M7 M8 M9 M10M11 0 0.193 0.196 0.202 0.184 0.182 0.182 0.184 0.185 0.18 0.172 0.1750.174 0 0.19 0.182 0.177 0.175 0.171 0.171 0.173 0.17 0.168 0.164 0.1620.163 10 ng/ml 0.259 0.328 0.204 0.267 0.199 0.286 0.255 0.218 0.2930.166 0.167 0.22 10 ng/ml 0.257 0.311 0.187 0.249 0.184 0.271 0.244 0.220.276 0.154 0.159 0.217 100 ng/ml 1.137 1.594 0.316 1.087 0.359 1.5131.093 0.785 1.468 0.164 0.164 0.884 100 ng/ml 1.111 1.553 0.299 1.0820.34 1.221 1.049 0.695 1.375 0.155 0.15 1.045 1 ug/ml 2.813 3.147 1.1793.147 1.375 2.877 3.053 2.703 3.253 0.199 0.171 3.053 1 ug/ml 2.6513.053 0.986 2.864 1.413 3.025 2.983 2.716 2.93 0.218 0.172 3.159 WT =wild type CTLA-4Fc. M1-M11 are CTLA-4Fc mutant proteins.

TABLE 3 Epitope mapping of chimeric L3D10 antibody. Binding to CTLA-4Fcproteins was performed by ELISA, with the amounts of biotinylatedprotein bound measured by horse-radish peroxidase (HRP)-conjugatedstreptavidin. Values shown are the OD450 measurements. Protein Conc. WTM1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 0 0.202 0.196 0.2 0.187 0.184 0.1890.192 0.198 0.187 0.179 0.179 0.183 0 0.195 0.187 0.185 0.18 0.176 0.1760.176 0.176 0.17 0.166 0.166 0.167 10 ng/ml 1.433 2.47 0.375 0.62 0.5071.539 1.033 0.714 1.233 0.18 0.18 0.202 10 ng/ml 1.518 2.432 0.317 0.5870.356 1.366 0.976 0.738 1.237 0.171 0.169 0.203 100 ng/ml 3.053 3.2531.384 2.318 2.142 2.841 2.699 2.495 2.909 0.295 0.215 0.635 100 ng/ml3.025 3.2.39 1.164 2.354 1.409 2.991 2.771 2.483 2.841 0.304 0.216 0.7591 ug/ml 3.373 3.268 2.387 3.184 2.651 3.025 3.092 3.147 3.136 0.9160.804 2.841 1 ug/ml 3.114 2.967 2.619 3.124 2.659 3.034 3.072 2.9913.034 0.916 0.868 2.983 WT = wild type CTLA-4Fc. M1-M11 are CTLA-4Fcmutant proteins

TABLE 4 Epitope mapping of humanized antibody mAb4. Binding to CTLA-4Fcproteins was performed by ELISA, with the amounts of biotinylatedprotein bound measured by horse-radish peroxidase (HRP)-conjugatedstreptavidin. Values shown are the OD450 measurements. Protein Conc. WTM1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 10 ng/ml 0.312 2.264 0.207 0.1980.194 0.407 0.22 0.194 0.247 0.177 0.181 0.172 10 ng/ml 0.29 2.297 0.1840.178 0.174 0.378 0.202 0.185 0.222 0.154 0.16 0.164 100 ng/ml 1.0772.827 0.203 0.27 0.219 1.371 0.459 0.281 0.725 0.171 0.17 0.172 100ng/ml 0.841 3.061 0.194 0.264 0.208 1.589 0.42 0.277 0.801 0.154 0.1550.159 1 ug/ml 2.51 2.881 0.339 0.882 0.473 2.79 1.992 1.169 2.33 0.1750.17 0.178 1 ug/ml 2.471 2.958 0.263 1.121 0.573 2.795 2.016 1.243 2.6420.167 0.169 0.185 WT = wild type CTLA-4Fc. M1-M11 are CTLA-4Fc mutantproteins

TABLE 5 Epitope mapping of humanized antibody mAb5. Binding to CTLA-4Fcproteins was performed by ELISA, with the amounts of biotinylatedprotein bound measured by horse-radish peroxidase (HRP)-conjugatedstreptavidin. Values shown are the OD450 measurements. Protein Conc. WTM1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 10 ng/ml 0.597 2.307 0.195 0.5440.189 1.239 0.603 0.19  0.5  0.373 0.169 0.157 10 ng/ml 0.535 2.2440.162 0.195 0.435 1.188 0.516 0.535 0.47  0.148 0.15  0.152 100 ng/ml1.947 2.632 0.182 0.389 0.248 2.601 1.296 0.521 2.001 0.15  0.15  0.152100 ng/ml 2.229 2.186 0.175 0.364 0.221 2.425 0.875 0.405 2    0.1370.139 0.148 1 ug/ml 2.724 2.05  0.259 1.662 0.725 2.654 2.355 1.4182.548 0.157 0.151 0.162 1 ug/ml 2.742 2.297 0.274 1.549 0.724 2.84 2.374 1.369 2.69  0.147 0.143 0.165 WT = wild type CTLA-4Fc. M1-M11 areCTLA-4Fc mutant proteins

TABLE 6 Raw data from a repeat study showing specific loss of antigenicepitope only in M11. As in Tables 2-5, except that additional controlswere included to shown specificity of the binding. Ab Conc hCTLA4-Fc M1M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 Biotin-LD310 0 0.18 0.187 0.377 0.1830.22 0.177 0.183 0.186 0.368 0.16 0.215 0.171 0 0.177 0.222 0.538 0.1670.18 0.229 0.177 0.142 0.217 0.293 0.114 0.155 10 ng/ml 1.706 2.6920.469 0.623 0.817 1.853 1.244 0.837 1.27 0.158 0.169 0.19 10 ng/ml 1.7992.779 0.333 0.593 0.563 1.802 1.331 0.884 1.454 0.213 0.157 0.194 100ng/ml 3.316 3.195 1.313 2.244 2.233 3.251 3.032 2.672 3.015 0.419 0.260.752 100 ng/ml 3.458 3.567 1.37 2.635 2.356 3.316 3.032 2.875 3.1570.346 0.272 0.746 1000 ng/ml 3.567 3.509 2.833 3.333 3.08 3.413 3.2823.299 3.352 1.124 0.945 2.888 1000 ng/ml 3.672 3.509 2.755 3.299 3.1453.537 3.316 3.352 3.435 1.161 0.941 2.914 Biotin-hB7-1 0 0.195 0.2 0.2020.193 0.192 0.197 0.195 0.196 0.192 0.186 0.185 0.186 0 0.192 0.1850.181 0.192 0.178 0.178 0.178 0.187 0.173 0.169 0.168 0.161 10 ng/ml0.316 0.37 0.216 0.304 0.22 0.345 0.279 0.258 0.326 0.177 0.176 0.239 10ng/ml 0.31 0.356 0.21 0.414 0.26 0.331 0.279 0.253 0.297 0.159 0.1870.236 100 ng/ml 1.581 1.882 0.333 1.245 0.527 1.813 1.235 0.899 1.5570.176 0.172 1.092 100 ng/ml 1.625 1.928 0.323 1.345 0.489 1.735 1.3850.987 1.643 0.162 0.155 1.283 1000 ng/ml 3.76 3.6 1.167 3.435 1.9733.316 3.413 3.101 3.635 0.232 0.185 3.568 1000 ng/ml 3.6 3.673 1.3163.51 2.009 3.459 3.413 3.183 3.635 0.215 0.181 3.673 Biotin-mAb4 10ng/ml 0.451 2.812 0.207 0.202 0.194 0.626 0.23 0.207 0.327 0.197 0.2050.181 10 ng/ml 0.417 2.693 0.181 0.179 0.177 0.642 0.22 0.195 0.32 0.1580.182 0.162 100 ng/ml 1.868 3.566 0.212 0.29 0.256 2.618 0.589 0.3451.532 0.172 0.174 0.171 100 ng/ml 1.938 3.317 0.203 0.274 0.247 2.1260.571 0.305 1.419 0.155 0.155 0.162 1000 ng/ml 2.99 3.568 0.268 1.1810.712 2.922 2.187 1.329 2.817 0.181 0.17 0.177 1000 ng/ml 3.033 3.510.266 1.184 0.759 3.071 2.358 1.475 2.869 0.144 0.171 0.187 Biotin-mAb510 ng/ml 0.983 2.654 0.202 0.218 0.197 1.409 0.429 0.218 0.727 0.1760.176 0.17 10 ng/ml 0.955 2.604 0.184 0.2 0.168 1.359 0.389 0.21 0.7610.148 0.154 0.152 100 ng/ml 2.669 3.007 0.232 0.534 0.319 2.908 1.8390.523 2.669 0.145 0.161 0.16 100 ng/ml 2.741 3.158 0.203 0.554 0.3742.895 1.741 0.478 2.604 0.145 0.148 0.157 1000 ng/ml 3.183 3.146 0.3271.837 1.019 2.966 2.817 1.72 3.042 0.173 0.163 0.174 1000 ng/ml 3.2093.316 0.321 1.867 1.015 3.196 2.857 1.766 3.051 0.143 0.163 0.187Biotin-L3D10 Biotin-L3D10 Biotin-hB7-1 Biotin-hB7-1 Ab conc mCTLA4-FchIg-Fc mCTLA4-Fc hIg-Fc Biotin-HL12 Biotin-HL12 Biotin-HL32 0 0.19 0.1980.202 0.191 0 0.189 0.184 0.18 0.185 10 ng/ml 0.201 0.201 0.338 0.1810.179 0.188 0.185 0.179 10 ng/ml 0.18 0.182 0.318 0.164 0.165 0.162 0.170.181 100 ng/ml 0.303 0.315 1.635 0.176 0.171 0.177 0.185 0.176 100ng/ml 0.314 0.326 1.668 0.165 0.162 0.163 0.165 0.171 1000 ng/ml 0.9421.385 3.569 0.18 0.177 0.182 0.184 0.183 1000 ng/ml 0.94 1.475 3.3530.179 0.172 0.177 0.176 0.187 mCTLA4 hIgG mCTLA4 hIgG mCTLA4 hIgG mCTLA4hIgG Biotin-L3D10 Biotin-hB7-1 Biotin-HL12 Biotin-HL32

Since L3D10 retained significant binding the M11, we tested if thebinding is specific. We coated plate with human CTLA-4-Fc (hCTLA-4Fc)mouse CTLA-4-Fc (mCTLA-4-Fc), Control IgG1-Fc or all mutant hCTLA-4-Fcand measured their binding to B7-1Fc along with L3D10, mAb4 and mAb5.The bulk of the data are presented. in Table 6. As shown in FIG. 17,biotinylated B7-1 binds hCTLA-4, mCTLA-4 and M11, equally well. Thespecificity of the assay is demonstrated by lack of binding to IgG1-Fc.Interesting, while L3D10-binding to M11 is stronger than those toIgG1-Fc and mCTLA-4-Fc, significant binding to IgG1-Fc suggest that thechimeric antibody binding to M11 maybe nonspecific. In contrast, none ofthe humanized antibodies bind to M11, mCTLA-4, and IgG1-Fc control.These data demonstrate that mutations introduced in M11 selectivelyablated L3D10, mAb4 and mAb5 binding to CTLA-4.

Using known complex structure 133, we mapped the CTLA-4 epitope in a 3-Dstructure. As shown in FIG. 18, the epitope recognized by these mAbslocalized within the area covered by B7-1. As such, L3D10, mAb4 and mAb5binding to CTLA-4 would be mutually exclusive to that of B7-1. The poorblocking of mAb4 and mAb5 is due to lower avidity rather thandistinctive binding domains.

Using a number of mutants of the human CTLA-4Fc protein in whichclusters of amino acids from the human CTLA-4 protein were replaced withamino acids from the murine Ctla-4 protein, we clearly demonstrate thatwhen we replace 4 amino acids that immediately follow the known B7-1binding domain of CTLA-4, MYPPPY, dose-dependent binding of theantibodies is largely abolished. The fact that the binding epitope mapsdirectly adjacent to the B7-1 binding domain correlates well with thedemonstrated ability of the L3D10 antibodies to block B7-CTLA-4interactions both in vitro and in vivo.

Example 7 Generation of Fusion Proteins that Show Diminished Binding toOne or More Cancer Immunotherapeutic Anti-CTLA-4 mAbs

We have generated a panel of 17 CTLA-4-Fc fusion proteins with variousmutations, designated M1-M17 (SEQ ID NOS: 7-23), which include the 11proteins identified in Example 10 (FIG. 16) plus 6 additional mutants(FIG. 19). Further mutations were made to M17. The new mutants arecalled M17-1, M17-2, M17-3 and M17-4 (SEQ ID NOS: 42-45). Thesemutations were generated anticipating that they would ablate binding toa broad spectrum of anti-CTLA-4 antibodies but retain binding to theCTLA-4 ligands, CD80 (B7-1) and CD86 (B7-2). As the first step, wecoated a given concentration of the fusion proteins on the plate andadded biotinylated B7-1Fc. As shown in FIGS. 20A and B, all but 2 fusionproteins (M9 and M10) retain binding to B7-1, although a significantreduction in B7-1 binding was found in M2, M4 and M7. Since theseproteins are functionally less active, it is unlikely that these mutantsare optimal for in vivo protection against autoimmune adverse effects.Additional data demonstrate that M15, M17, M17-1 and M17-2 retainedstrong binding to both CD80 and CD86 (FIG. 21).

Next, we evaluated if these mutant proteins (M1-M17, and M17-1 to M17-4)have lost binding to anti-CTLA4 mAbs that are either approved forclinical use or being developed for clinical therapy. A total of 4 mAbswere tested. For mAb1 (Ipilimumab), fusion proteins M11, M13, M15, M17,M17-1, M17-2 and M17-3 have lost binding, and thus can be used forprotection against autoimmune diseases induced by this mAb (FIG. 22A).For autoimmune diseases induced by mAb2 (Tremelimumab), M15, M17, M17-2and M17-3 will be effective (FIG. 22B), For mAb4 and mAb5, M11, M12,M14, and M16 are likely the most effective, although M3 and M17 may alsowork (FIGS. 23, 24 and 25).

Therefore, as summarized in Table 7, CTLA4-Fc fusion protein M17 hasbroad spectrum activity for use in protection against autoimmunediseases induced by anti-CTLA4 antibodies. M17-3 shows low binding toanti-CTLA4 antibodies but low binding to B7-1 and 137-2. However, M17-2showed better binding to B7-1 and B7-2 and yet shows acceptably lowcross-reactivity against a broad range of anti-human CTLA-4 mAbs.

TABLE 7 Identification of CTLA4-Fc fusion proteins that retains bindingto B7-1, but not to anti-CTLA4 mAb. Relative binding is presented by “+”or “−”, where “++++” is the strongest binding and “−” indicates nobinding. CTLA4-Fc Fusion mAb1 mAb2 protein hB7-1Fc hB7-2Fc (Ipilimumab)(Tremelimumab) mAb4 mAb5 abatacept +++ ND +++ +++ +++ +++ hIgGFc ctl − −− − − − M1 +++ ND +++ ND ++++ ++++ M2 + ND + ND − − M3 +++ ND +++ ND + +M4 + ND + ND + + M5 +++ ND +++ ND +++ +++ M6 +++ ND +++ ND + ++ M7 ++ND + ND + + M8 +++ ND +++ ND +++ +++ M9 − ND − ND − − M10 − ND − ND − −M11 +++ ND − +++ − − M12 +++ ND + +++ − − M13 +++ ND − +++ ++ ++ M14 +++ND + +++ − − M15 +++ +++ − − +++ ++++ M16 +++ ND + +++ − − M17 +++ ++ −− + + M17-1 +++ ++ − +++ ++ − M17-2 +++ +++ − − +/− − M17-3 ++ +/− − − −− M17-4 +++ + + +++ ++ − ND = not determined.

Example 8 Blocking B7-1 and B7-2 does not Present ImmunotherapeuticEffect of Ipilimumab

A critical requirement for treating anti-CTLA-4-immunothery relatedadverse effects with CTLA-4-Fc mutants that bind to B7-1/B7-2 is thatblocking B7-1 and B7-2 does not affect the immunotherapeutic effect.Since mice with targeted mutations of Cd80 (encoding B7-1) and Cc186(encoding B7-2) do not have Treg (17) and thus express very littleCtla4, we tested this prediction by using a saturating dose of anti-B7-1(1G10) and anti-B7-2 (GL-1) mAbs, which block the binding of humanCTLA-4 to mB7-1 and mB7-2, respectively (FIG. 26A). As diagrammed inFIG. 26B, we treated MC38 tumor-bearing mice with Ipilimumab inconjunction with either control Ig or a combination of anti-mB7-1 andanti-mB7-2 mAbs. The anti-mB7 mAbs used completely masked all B7-1 andB7-2 in the PBL as their binding to new anti-mB7 mAb is reduced to whatwas observed in mice with targeted mutations of both Cd80 and Cd86 (dKO)(FIG. 26C, D). Functional blockade of B7 by anti-B7-1 and anti-B7-2 mAbswas evaluated using sera collected at day 22 after tumor challenge toevaluate anti-human IgG antibody response and confirmed by the fact thatantibody response against Ipilimumab was abrogated (FIG. 26E).Importantly, saturating blockade of mB7 did not affect theIpilimumab-induced tumor rejection as anti-mB7 and control Ig treatedmice are equally responsive to Ipilimumab (FIG. 26F). These dataindicate that anti-B7-1 and anti-B7-2 as well as B7-binding CTLA-4fusion proteins that do not bind anti-CTLA-4 antibodies can be used totreat irAE associated with anti-CTLA-4 immunotherapy, while leaving theprotective immunotherapeutic activity intact.

Example 9 Use of CTLA-4 Fusion Proteins to Treat Autoimmune AdverseEffects Associated with Anti-CTLA-4 and Anti-PD-1 Combination Therapy

Combination therapy with anti-CTLA-4 and anti-PD-1 represents the mosteffective cancer immunotherapy. However, with greater than 50% ofpatients receiving immunotherapy developing irAEs. there is an urgentneed to develop therapeutic approaches to treat or prevent irAE. Asdemonstrated in Example 8, the immunotherapeutic effect of Ipilimumab(mAb1) is unaffected by the blockade of B7-1 and B7-2. These findingsprompted us to test if mutant CTLA-4FC can be used to treat irAEassociated with Ipilimumab+anti-PD-1 combination therapy. SinceIpilimumab does not bind to CTLA-4-Fc fusion proteins M11 or M15(belatacept), we tested to see if co-administration of belatacept or M11or M15 can ameliorate irAE, using retarded growth as a readout of irAE(see Example 2). As shown in FIG. 27, low doses of either M11 (100μg/injection) or M15 (200 μg/injection) prevented irAE. In addition toM11 and M15, other proteins with similar properties, including M13, M16and M17 may also be used.

Example 10 Identification of Fusion Proteins that do not Interfere withAnti-CTLA4 Immunotherapeutic Activity

In order to determine whether CTLA-4-Fc fusion proteins interfere withanti-CTLA4 immunotherapeutic effects, we injected MC38-tumor bearingmice with either control IgG Fc (200 μg/injection) or Ipilimumab (100μg/injection) in combination with control IgGFc (100 μg/injection) orthe CTLA4 fusion proteins, Abetacept, M15 or M17 (100 μg/injection) ondays 7, 10, 13 and 17. The tumor growth rate were shown in FIG. 28A,while the mouse survival to early removal endpoint are shown in FIG.28B. As expected, Abatacept ablated the immunotherapeutic effect of theIpilimumab. In contrast, M15 and M17 did not interfere with thetherapeutic effect of Ipilimumab.

Example 11 Modulation of T Cell Activation and its Associated Adverseeffect in NSG Mice Reconstituted with Human Hematopoietic System

To determine the impact of CTLA-4 fusion proteins on the activation ofhuman T cells and associated irAE in vivo, we reconstituted the 3 weekold NSG mice with human hematopoetic stem cells and monitored T cellactivation. As shown in FIGS. 29-32, different CTLA-4 mAbs appear tohave different effects on T cell phenotype and the adverse effects ofIpilimumab. Thus, as shown in. FIG. 29A, treatment with Ipilimumabprevents NSG mice from gaining weight, and this is prevented byco-administration of M15 or M17-2. Interestingly, M15 induced elevationof Alanine transaminase when used in conjunction with Ipilimumab (FIG.29B). These data suggest that M15 may induce liver damage when used inconjunction with Ipilimumab. Importantly, no such effect was observedwhen M17-2 was used in conjunction with M15.

To understand the immunological basis of the differentiation between M15and M17, we sacrificed the NSG mice on day 31 and analyzed thecomposition of T cell subsets. As shown in FIG. 30, M17 induced morecentral memory T cells, while reducing effector T cells. The reductionof effector T cells may explain the reduced adverse effect as this isthe subset that migrates into organs to cause immune destruction.Furthermore, since NKT cells are the primary mediator of hepatitis, wecompared M15 and M17-2 for their impact on NKT cells, based onexpression of both CD56 and CD3. As shown in FIG. 31, M17-2 reduced thenumber of NKT while M15 enhanced it.

The regulatory T cells suppress autoimmune diseases and cancer immunity.It is therefore desirable to reduce Treg to enhance cancer immunity.However, if the reduction is too severe, one may induce autoimmunedisease. Consistent with a critical role for B7-CD28 interactions in thegeneration and maintenance of Treg in mice, both M15 and M17-2significantly reduced the of Treg (FIGS. 32A and B). However, M15 ismuch more effect in reducing Treg (FIGS. 32A and B). Furthermore, Tregfrom M15-treated mice had substantially lower CTLA-4 (FIGS. 32C and D).Given the essential role for CTLA-4 in Treg function in vivo, it islikely that Treg from the M15-treated mice are functionally impaired.Thus, a severe reduction of Treg combined with selective reduction ofCTLA-4 protein levels suggest an intriguing possibility that M15 mayinduce autoimmune destruction when used in conjunction Ipilimumab.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

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1.-25. (canceled)
 26. A method of treating adverse events associatedwith cancer immunotherapy in a subject in need thereof, comprisingadministering to the subject a CTLA-4 protein, wherein the CTLA-4protein comprises an extracellular domain of CTLA-4 comprising thesequence set forth in SEQ ID NO:
 38. 27. The method of claim 26, whereinthe CTLA-4 protein binds to at least one of B7-1 and B7-2.
 28. Themethod of claim 27, wherein the CTLA-4 protein does not block the cancerimmunotherapeutic effects of an anti-CTLA-4 antibody.
 29. The method ofclaim 28, wherein the anti-CTLA-4 antibody is Ipilimumab.
 30. The methodof claim 26, wherein the extracellular domain of CTLA-4 is fused at itsC-terminus to a Fc region of a human immunoglobulin protein selectedfrom the group consisting of IgG1, IgG4, and IgM.
 31. The method ofclaim 30, wherein the CTLA-4 protein comprises the sequence set forth inSEQ ID NO:
 21. 32. The method of claim 26, wherein the CTLA-4 protein ispresent at a therapeutically effective amount in a pharmaceuticalcomposition comprising a physiologically acceptable carrier orexcipient.
 33. The method of claim 26, wherein the cancer immunotherapycomprises an anti-CTLA-4 antibody.
 34. The method of claim 33, whereinthe anti-CTLA-4 antibody is Ipilimumab.
 35. The method of claim 34,wherein the cancer immunotherapy further comprises an anti-PD-1antibody.
 36. The method of claim 26, wherein the subject has cancer.37. The method of claim 31, wherein the subject has cancer.
 38. Themethod of claim 33, wherein the subject has cancer.
 39. A method oftreating adverse events associated with cancer immunotherapy in asubject in need thereof, comprising administering to the subject aCTLA-4 protein, wherein the amino acid sequence of the CTLA-4 proteinconsists of the sequence set forth in SEQ ID NO:
 21. 40. The method ofclaim 39, wherein the CTLA-4 protein is present at a therapeuticallyeffective amount in a pharmaceutical composition comprising aphysiologically acceptable carrier or excipient.
 41. The method of claim39, wherein the cancer immunotherapy comprises an anti-CTLA-4 antibody.42. The method of claim 41, wherein the anti-CTLA-4 antibody isIpilimumab.
 43. The method of claim 42, wherein the cancer immunotherapyfurther comprises an anti-PD-1 antibody.
 44. The method of claim 39,wherein the subject has cancer.
 45. The method of claim 41, wherein thesubject has cancer.